|Year : 2003 | Volume
| Issue : 1 | Page : 37-42
In vitro activity of norfloxacin against uropathogens and drug efficacy in simulated bladder model under diabetic conditions
H Anandkumar , A Dayanand , CS Vinodkumar , I Kapur
Department of Microbiology, KBN Institute of Medical Sciences, Gulbarga - 585 104, Karnataka, India
Department of Microbiology, KBN Institute of Medical Sciences, Gulbarga - 585 104, Karnataka, India
PURPOSE: The in vitro activity of norfloxacin was determined to maximize the correlation between susceptibility testing of the drug and the results of clinical therapy of urinary tract infection in diabetics. This study was carried out to observe the effect of changing concentration of norfloxacin on the growth of uropathogens under diabetic conditions. METHODS: The standard broth microdilution method was carried out to determine the minimum inhibitory concentration (MIC) using Mueller Hinton broth by varying pH of the medium (5.0, 5.5, 6.0, 6.5 and 7.0) and glucose concentration (100, 250, 500, 1000 and 2000 mg/dL). A specially designed mechanical bladder model system simulating hydrokinetic conditions that exist in the urinary tract of diabetics was employed. RESULTS: The loss of activity of norfloxacin was more pronounced (> four folds) at pH 5.0 and 2000 mg/dL sugar concentration. These findings were consistent with the experiment 'in vitro simulated bladder model' by exposing bacterial growth to varied norfloxacin and sugar concentration. CONCLUSIONS: Although norfloxacin is a drug of choice for non-diabetic and diabetic individuals with mild to moderate glucosuria, in severe diabetic individuals norfloxacin may not be an effective drug.
|How to cite this article:|
Anandkumar H, Dayanand A, Vinodkumar C S, Kapur I. In vitro activity of norfloxacin against uropathogens and drug efficacy in simulated bladder model under diabetic conditions. Indian J Med Microbiol 2003;21:37-42
|How to cite this URL:|
Anandkumar H, Dayanand A, Vinodkumar C S, Kapur I. In vitro activity of norfloxacin against uropathogens and drug efficacy in simulated bladder model under diabetic conditions. Indian J Med Microbiol [serial online] 2003 [cited 2020 Jan 19];21:37-42. Available from: http://www.ijmm.org/text.asp?2003/21/1/37/8313
Diabetic individuals are more susceptible to urinary tract infections (UTIs) than non diabetic subjects., The following factors have been suggested as possible causes (i) neutrophil function impaired; (ii) glucose in urine supports bacterial growth better; and (iii) adherence to uroepithelial cells is increased. It has been established that uropathogenic bacteria grow well in human urine, whereas non uropathogenic bacteria do not. It has also been suggested that not only pH and osmolality, but also glucosuria enhance bacterial growth. In 1961, O'Sullivan et al compared the growth of E.coli (from patients with asymptomatic bacteriuria) in urine with and without the addition of glucose 2% (200 mg/dL moderate glucosuria). In a recent study, 50% of diabetic patients with positive urine culture were found to have glucosuria in the range of 300-1000 mg/dL. The activity of quinolones (fleroxacin and norfloxacin) was found to be uniformly two to four fold lowered in urine than in broth or serum at pH 7.0. Although the presence of diabetes is a definite risk factor for urinary tract infection, we attempted to determine the host factor for higher susceptibility to urinary tract infection in diabetes. The fluoroquinolones are known to have well established high degrees of efficacy in various types of UTIs. In comparison with other antibiotics, the spectra of activity are superior. In this study, we used a fluoroquinolone (norfloxacin) to study its efficacy on growth of uropathogens in presence of diabetes, using simulated bladder model.
| ~ Material and Methods|| |
A total of 135 diabetic patients showing symptoms of urinary tract infection were screened for significant bacteriuria. The mid-stream urine samples collected from all the patients were transported to the laboratory within half to one hour. In the laboratory the specimens were examined for pus cells, red blood cells and casts. A standard loop technique was used to place 0.01 mL of urine on MacConkey's agar and blood agar. The plates were examined after overnight incubation at 37°C to quantify the organisms grown. The colony count was done and organisms were identified by conventional methods. Microbroth dilution method was employed to determine minimum inhibitory concentration (MIC) of confirmed clinical isolates against the drug norfloxacin (Cipla Pharma Ltd.).
MIC of norfloxacin was determined in Mueller Hinton Broth. Norfloxacin was dissolved in methanol and 1N NaOH and diluted with distilled water just before use. Care was taken to avoid unnecessary exposure of norfloxacin solution to day light. Final concentration of norfloxacin in broth ranged from 128 to 0.008 µg/mL. Drug free control plates were included throughout the study. The pH of medium was measured with a pH meter (Systronics), pH was adjusted with 1N HCl or 1N NaOH ranging from 5.0 to 7.0 (5.0, 5.5, 6.0, 6.5, 7.0). The required range (100, 250, 500, 1000 and 2000 mg/dL) of sugar concentration in correlation with glucosuria of diabetic cases was obtained by adding glucose (HiMedia).
In vitro static turbidometry method at different time intervals was employed to assess the antibacterial activity of norfloxacin by observing the growth rate of bacteria.  mL of Mueller Hinton broth were inoculated with uropathogenic isolate from an overnight broth culture, adjusted with 0.5 McFarland standard to achieve an inoculum size of 105 cfu/mL. MIC concentration of norfloxacin was added in the broth and incubated at 37ºC for 18 hours. The opacity of the broth was measured (by TRACE-20 semi autoanalyzer) at every one hour interval during incubation.
A mechanical bladder model system simulating hydrokinetic conditions that exist in diabetics was employed. The working protocol of bladder model system is depicted in [Table - 1]. Twenty millilitre of inoculum matching 0.5 McFarland standard was prepared to achieve the inoculum size 105 cfu/mL from uropathogens and was added in a sterile empty glucose bottle placed up right with rubber stopper and incubated at 37ºC. A known concentration of norfloxacin was added into the flask containing 20 mL culture.
The dilution rate was adjusted to 20mL/hr. After 3 hours the norfloxacin was withdrawn and the broth was used for further dilution as indicated in the protocol. The optical density of the broth collected after every 3 hour was measured at 540nm and recorded.
| ~ Results|| |
Of total 135 cases of symptomatic urine samples from diabetic cases with glucosuria analyzed, 102 cases (75.6%) showed significant bacteriuria. E.coli (47.1%) was isolated as a predominant uropathogen followed by Klebsiella pneumoniae (27.5%), Pseudomonas aeruginosa 5.7%), Staphylococcus saprophyticus (4.9%), Staphylococcus aureus (2.9%), Proteus mirabilis (0.9%) and Enterococcus faecalis ).
The influence of pH and sugar on MIC of norfloxacin against uropathogens is depicted in [Table - 2]. The MIC of all the clinical isolates used were two to four fold higher in 1000 and 2000 mg/dL sugar than moderate (250-1000 mg/dL) and mild (100-250 mg/dL) glucosuria levels. With the combined effects of low pH (5.0 to 5.5) and higher sugar concentration (1.5 to 2.0%), norfloxacin activity was reduced more than four folds.
The effect of norfloxacin and sugar concentration on the growth of uropathogens exposed at different time intervals in simulated bladder model is depicted in [Table - 3]. In presence of initial concentration of 300 mg/mL norfloxacin E.coli showed no growth but bacterial growth reemerged after 9 hours at 2000 mg/dL and after 12 hours incubation at 1000 and 2000 mg/dL. Klebsiella pneumoniae showed growth after 12 hours of incubation at 1000 and 2000 mg/dL sugar, Proteus mirabilis did not show any growth even after 12 hours of incubation. The growth of Pseudomonas aeruginosa was observed after 6 hours at >500 mg/dL sugar. Staphyloccocus aureus showed growth after 12 hours of incubation in presence of >100 mg/dL sugar.
[Figure - 1], [Figure - 2], [Figure - 3], [Figure:4] indicate the influence of sugar and norfloxacin on growth of uropathogenic E.coli obtained from symptomatic diabetic UTI cases at varied pH by static turbidometry. The growth of E.coli appeared in presence of norfloxacin (0.06 g/mL) after 5 hours of incubation at pH 5.0 in all ranges and the maximum growth was attained at 1000 and 2000 mg/dL sugar and pH 6.0.
| ~ Discussion|| |
The in vitro activity of norfloxacin was determined to maximize the correlation between susceptibility testing of the drug and the results of clinical therapy of urinary tract infection in diabetes. Generally, glucosuria of 0.0 to 6.0 mg/dL of urine is accepted as normal, 100 to 250 mg/dL as mild diabetes, and 250 to 1000 mg/dL as severe diabeties.
MIC of norfloxacin at the sugar concentration of 1000 and 2000 mg/dL were 2-4 folds higher than mild and moderate glucosuria levels. But the combined effect of pH and sugar had greater impact on norfloxacin activity. At higher sugar concentration (1.5 to 2.0%) and low pH (5.0 to 5.5), norfloxacin activity was reduced more than four folds. Therefore, it clearly reveals that the efficacy of norfloxacin is very high at normal (non-diabetic) conditions of urine pH and sugar than at the altered condition of urine pH and sugar concentrations. These findings are similar to the observations made by Haul and Felber who recorded the higher loss of activity of norfloxacin in urine at pH 5.0 compared to 6.0 and 7.0. Chin et al state that loss of activity of norfloxacin is more pronounced in urine attributed to magnesium content to broth and serum.
The effect of norfloxacin on the growth of uropathogen at varied pH and sugar concentration was found to be efficient up to 10-12 hours of incubation at pH 7 and mild to moderate glucose concentration. In an earlier study, Jackson and Grieble16 had shown that lower concentration of glucose (0.1%; 10 mg/dL) had no effect on bacterial yield, although the growth rate increased. But in higher concentration (1000 and 2000 mg/dL) even at pH 7.0 growth of bacteria appeared as early as 7 to 9 hours of incubation in many instances. In a recent study by Balasoiu et al, 50% of diabetic patients with positive urine culture were found to have glucosuria in the range of 300-1000 mg/dL. The uniform reduction in norfloxacin activity was observed in reduced pH and reached lowest at pH 5.0. The bacterial growth at pH 5.0 appeared as early as 2-3 hours of incubation in presence of norfloxacin. The combined effect of low pH and high sugar concentration has adverse impact on norfloxacin efficacy.
In the simulated bladder condition, all the bacterial strains used were inactive after 3 hours of incubation at the initial concentration of 300 g/mL but E.coli showed reemergence of growth after 9 hours of incubation at 2000 mg/dL sugar, while Pseudomonas aeruginosa growth was observed as early as 6 hours of incubation at 1000 mg/dL sugar. Bacterial growth observed after 12 hours of incubation has no clinical significance as the patient is administered the second dose of norfloxacin (twice daily regimen).
These in vitro results may not exactly reflect the in vivo condition as the human urinary tract is a dynamic system in which fresh urine is constantly being formed and normal micturition occurs at every 4-6 hours. This urine flow is also important in the defense against infection. Further more, the urine pH and osmolality change over time. For these reasons, the low urinary pH, as a result of bacterial metabolism probably does not play an important role as an inhibitor of further bacterial growth in vitro and in the clinical situation very high glucose concentration probably enhances bacterial growth. This may explain greater susceptibility of diabetics to urinary tract infection.
To conclude, norfloxacin may be used in non-diabetic and diabetic individuals with mild to moderate glucosuria. However, in severe diabetic individuals, norfloxacin may not be a drug of choice.
| ~ Acknowledgement|| |
The authors would like to thank Dr Sirajuddin, Dr Nasir Bin Ali, and KBN Teaching & General Hospital, Gulbarga, for their help in carrying out this work.
| ~ References|| |
|1.||Hansen RO. Bacteriuria in diabetic and non-diabetic out patients. Acta Medica Scandinavian 1964;176:721-730. |
|2.||Patternson JE, Andriole VT. Bacterial urinary tract infections in diabetes. Infect Dis Clin North Am 1995;9:25-51. |
|3.||Hoepelman IM. Urinary tract infection in patients with diabetes mellitus. Int J Antimicrobial Agents 1994;4:113-116. |
|4.||Stamey TA, Mihard G. Observations on the growth of urethral and vaginal bacteria in sterile urine. J Urol 1980;124:461-463. |
|5.||Asscher AW, Sussman M, Waters WE, Harwad Davis R, Chick S. Urine as a medium for bacterial growth. Lancet 1966;2:1037-1041. |
|6.||O'Sullivan DJ, Fitz Gerald MG, Meynell MJ, Malin SJM. Urinary tract infection: A comparative study in the diabetic and general populations. BMJ 1961;1:786-788. |
|7.||Balasoiu D, Van Kessel KC, Van Kats-Renand HJ, Collect TJ, Hoepelman AI. Granulocyte function in women with diabetes and asymptomatic bacteriuria. Diabetes Care 1997;20:392-395. |
|8.||Chin NX, Brittain DC, Nen HC. In-vitro activity of R0 23-6240, a new fluorinated 4-quinolone. Antimicrob agents Chemother 1986;29:675-80. |
|9.||Norrby SR. Treatment of urinary tract infections with quinolone antimicrobial agents. Quinolone Antimicrobial Agents, 2nd Edn., (Ed) Hooper DC (American Society of Microbiology, Washington DC), 1993. |
|10.||CVS Kumar, A Jairam, S Chetan, P Sudesh, I Kapur, Srikaramallya. Asymptomatic bacteriuria in school going children. Indian J Med Microbiol, 2002;20:29-32. |
|11.||National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically approved standards. 4th Ed. NCCLS document M7-A5. NCCLS, 940 West Valley Road, Wayne, Pennsylvania, USA, 1993. |
|12.||Hou LP, Felber AM. Effects of method, medium, pH and inoculum on the in-vitro antibacterial activities of fleroxacin and norfloxacin. J Antimicrob Chemother 1988;22:Suppl. D:71-80. |
|13.||Collee JG, Duguid JP, Fraser AG, Marmion BP. In: Mackie & McCartney Practical Medical Microbiology, 14th Ed. (Churchill Livingstone, London), 1996:845-852. |
|14.||Greenword D, Osman M, Goodwin J, Slack R. J Antimicrob Chemother 1984;13:315-323. |
|15.||Fine J. Glucose content of normal urine. BMJ 1965;1:1209-1214. |
|16.||Jackson GG, Grieble HG. Urinary tract infection: Pathogenesis of renal infection. Arch Intern Med 1957;100:692-700. |